Integrin α1 subunit is up-regulated in colorectal cancer

Abstract

Background: Colorectal cancer remains one of the leading causes of death from cancer in industrialized countries. Integrins are a family of heterodimeric glycoproteins involved in bidirectional cell signaling and participate in the regulation of cell shape, adhesion, migration, differentiation, gene transcription, survival and proliferation. The α1 subunit is known to be involved in RAS/ERK proliferative pathway activation and plays an important role in mammary carcinoma cell proliferation and migration. In the small intestine, α1 is present in the crypt proliferative compartment and absent in the villus, but nothing is known about its expression in the colon mucosa, or in colorectal cancer.

Results: In the present study, we demonstrated that in the colon mucosa, α1 is present in the basolateral domain of the proliferative cells of the crypt, and in the surrounding myofibroblasts. We found higher levels of α1 mRNA in 86% of tumours compared to their corresponding matched margin tissues. Immunohistochemical analysis showed that α1 staining was moderate to high in 65% of tumour cells and 97% of the reactive cells surrounding the tumour cells vs 23% of normal epithelial cells.

Conclusion: Our findings suggest an active role for the α1β1 integrin in colorectal cancer progression.

Figure 1

Representative immunohistochemical images showing expression of the α1 integrin subunit in CRC (B, D, F, H, L) and corresponding matched resection margins (A, C, E, G, K). The α1 subunit was found to be expressed at higher levels in a significant number of CRC tumours (D, F, H) compared to their corresponding matched normal tissues (C, E, G) where it was found to be predominantly expressed in the proliferative cells of the crypt and below detection levels in normal surface epithelial cells (C, G). Note that the subepithelial myofibroblasts were also stained for α1 in both normal tissues (A, E, G) and tumours (B, D, F, H). Scores: The margin in A and the tumour in B were both scored 0 (negative) whereas the tumour in D was scored 2 (strong) compared to score 0 (weak) for the matched margin C. The tumour in F was scored 1 (moderate) as was its corresponding margin in E (moderate). The margin in G was scored 0 and the matched tumour in H was scored 2. To validate the specificity of the primary goat anti-α1 antibody, adjacent sections of the same normal (I, K) and cancer (J, L) specimens were stained using 5 μg/ml of non-immune IgG (I, J) or anti-α1 IgG (K, L) as primary antibody. Scale bars = 50 μm.

Figure 2

Representative indirect immunofluorescence images showing double-stained sections for the detection of the α1 integrin subunit and laminin in a normal colon mucosa (A and B) and a moderately differentiated colon adenocarcinoma specimen (C, D). The α1 subunit (red signal) was observed at the basolateral domain of normal epithelial cells of the lower crypt compartment (LC in panel A and E in panel B) compared to weak signal in the upper compartment (UC, panel A). In CRC, α1 was also localized at the basolateral domain of tumour epithelial cells (E) as well as in the adjacent subepithelial myofibroblasts (MF) (Panels C, D). Basement membrane (arrows) located at the interface between the two tissues was stained with an anti-laminin antibody (green staining). Nuclei were stained with DAPI (blue). Scale bar = 50 μm.

Figure 3

Expression of the α1 integrin subunit in CRC samples. (A) The α1 integrin subunit mRNA levels were evaluated by qPCR in 65 sets of colorectal cancers and corresponding resection matched margins. Transcript levels for α1 were found to be increased in tumours relative to their corresponding resection margins in 86% of samples (dotted line: cut off = 2). Expression of the α1 gene was calculated by the ΔΔCt method and normalized to B2M expression. (p < 0.001, One-Sample T Test). (B) To illustrate the individual tendency of paired samples, transcript levels of α1 were expressed as 1/ΔCt in each of the 65 matched margin and corresponding cancers showing the significant tendency for a higher level of expression in cancers (p < 0.001, t-test two-tailed). (C) However, α1 subunit mRNA levels did not correlate with tumour stage (stage 1; 8 patients, stage 2; 23 patients, stage 3; 26 patients and stage 4; 8 patients). (D) Integrin α1 expression at the protein level was analysed by immunohistochemistry on a TMA containing 65 colorectal tissues and their corresponding resection margins only considering epithelial staining. As illustrated in Figure 1, α1 immunostaining was scored as 0: negative or weak, 1: moderate or 2: strong staining. Results show that α1 staining in carcinoma vs normal epithelial cells from the matched controls was significantly higher (McNemar-Bowker’s test, p < 0.001) in 37 specimens (57%, gray area), similar in 25 specimens (38%) and lower in 3 specimens (5%). (E) The relative α1 integrin subunit expression was classified as negative/weak or moderate/strong. The results show that only 23% of the normal tissues displayed moderate/strong epithelial staining for α1 compared to 65% of cancer cells and 97% of the peri-tumoral stromal cells. Bars represent 95% confidence level.